Present treatments for cell proliferative diseases include surgery, treatment with cytotoxic agents, radiation, and combinations of the preceding. Treatment with cytotoxic agents, also referred to throughout this specification as antineoplastic agents or chemotherapeutic agents, often produces significant toxic side effects, including destruction of normal cells. Well-characterized toxicities include nausea and vomiting, myelosuppression, alopecia and mucosity. Serious cardiac problems are also associated with certain combinations of cytotoxic agents, e.g., doxorubicin and paclitaxel, but are less common.
Example classes of cytotoxic agents include, for example, the anthracycline family of drugs, the vinca alkaloids, the mitomycins, the bleomycins, the cytotoxic nucleosides, the taxanes, the epothilones, discodermolide, the pteridine family of drugs, diynenes and the podophyllotoxins. Members of those classes include, for example, doxorubicin, carminomycin, daunorubicin, aminopterin, methotrexate, methopterin, dichloromethotrexate, mitomycin C, porfiromycin, 5-fluorouracil, 6-mercaptopurine, gemcitabine, cytosine arabinoside, podophyllotoxin or podophyllotoxin derivatives, such as etoposide, etoposide phosphate or teniposide, melphalan, vinblastine, vincristine, leurosidine, vindesine, leurosine, paclitaxel and the like. Other useful antineoplastic agents include estramustine, cisplatin, carboplatin, cyclophosphamide, bleomycin, gemcitibine, ifosamide, melphalan, hexamethyl melamine, thiotepa, cytarabin, idatrexate, trimetrexate, dacarbazine, L-asparaginase, camptothecin, CPT-11, topotecan, ara-C, bicalutamide, flutamide, leuprolide, pyridobenzoindole derivatives, interferons and interleukins.
Taxanes and vinca alkaloid drugs are microtubule-affecting agents. Both taxanes and vinca alkaloids are based on naturally occurring compounds derived from plants. The taxanes are derived from the needles and twigs of the European yew, or the bark of the Pacific yew. Vinca alkaloids are derived from the periwinkle plant.
An example of a taxane is Taxol® (paclitaxel), which is a complex diterpene. Studies have shown that paclitaxel blocks and/or prolongs cells in the G2 phase of the cell cycle. The inability of paclitaxel-treated cells to pass through G2 of the cell cycle may result from paclitaxel binding to and stabilizing microtubules (Schiff, P. B. and Horwitz, S. B., 1980, Proc. Natl Acad. Sci. USA, 77:1561–1565). Stabilization of the microtubles prevents the normal reorganization of the microtubules necessary for interphase and mitotic functions (Wani et al., 1971, J. Am. Chem. Soc., 93, 2325–2327; Schiff, P. B., supra). Taxol® also blocks the migration behavior of cells in culture (reference).
Paclitaxel has been shown to possess cytotoxicity and antitumor activity against certain cancers, including those not effectively treated by other cancer treatments. Further, paclitaxel was approved by the FDA in 1992 for the treatment of advanced ovarian cancer and in 1994 for the treatment of breast cancer. Paclitaxel is currently in clinical trials for the treatment other cancers, including lung cancer. Additionally, paclitaxel has been reported to increase the sensitivity of cells to the effects of ionizing radiation (see, U.S. Pat. No. 6,080,777).
Another class of antineoplastic agents are platinum complexes. Examples of such complexes are cisplatin and carboplatin. Platinum complexes are thought to disrupt DNA function by binding to DNA. For example, cisplatin kills tumor cells via formation of covalent, cross- or intrastrand DNA adducts (Sherman et al., 1987, Chem. Rev., 87, 1153–81).
Platinum complexes are often associated with side effects. For example, Cisplatin treatment can result in renal toxicity. Additionally, Carboplatin treatment often results in hematologic toxicity. Both cisplatin and carboplatin can cause neurotoxic effects, and gastrointestinal distress, such as nausea and vomiting.
Yet another class of antineoplastic drugs are topoisomerase II inhibitors. At present the FDA has approved six antineoplastic drugs, which inhibit topoisomerase II. These drugs include doxorubicin, daunorubicin, idarubicin, mitoxantrone, etoposide, and anteniposide. Doxorubicin, idarubicin, and daunorubicin belong to the class of topoisomerase inhibitors known as anthracyclines. Mitoxantrone belongs to the class know as anthraquinone. Etoposide and teniposide belong to the class of compounds known as podophyllotoxin.
Anthracyclines antibiotics were initially isolated from fermentation products of Streptomyce peucetus. The most widely used anthracycline is doxorubicin. It is used in a wide variety of cancers, including lymphomas, breast cancer, sarcomas, Kaposi's sarcoma and leukemias. It is one of the primary drugs used for the treatment of breast cancer and soft tissue sarcomas.
Doxorubicin has serious side effects, including suppression of white blood cell and platelet formation. Doxorubicin can also cause heart damage due to the formation of free-radical intermediates which destroy myocardial cells. All patients who take doxorubicin suffer hair loss.
Another class of antineoplastic agents are the ansamycins. Ansamycin antibiotics are natural products derived from Streptomyces hygroscopicus that have profound effects on eukaryotic cells. The ansamycins were originally isolated on the basis of their ability to revert v-src transformed fibroblasts (Uehara, Y. et al., 1985, J. Cancer Res., 76: 672–675). Subsequently, they were shown to have antiproliferative effects on cells transformed with a number of oncogenes, particularly those encoding tyrosine kinases (Uehara, Y., et al., 1988, Virology, 164: 294–98). inhibition of cell growth is associated with apoptosis and, in certain cellular systems, with induction of differentiation (Vasilevskaya, A. et al., 1999, Cancer Res., 59: 3935–40). An ansamycin derivative, 17-allylamino 17-demethoxygeldanamycin (17-AAG), is currently in phase I clinical trials. The use of ansamycins as anticancer agents are described in U.S. Pat. Nos. 4,261,989, 5,387,584 and 5,932,566. The preparation of the ansamycin, geldanamycin, is described in U.S. Pat. No. 3,595,955 (incorporated herein by reference).
The eukaryotic heat shock protein 90s (HSP90s) are ubiquitous chaperone proteins, which bind and hydrolyze ATP. The role of HSP90s in cellular functions are not completely understood, but recent studies indicate that HSP90s are involved in folding, activation and assembly of a wide range of proteins, including key proteins involved in signal transduction, cell cycle control and transcriptional regulation. For example, researchers have reported that HSP90 chaperone proteins are associated with important signaling proteins, such as steroid hormone receptors and protein kinases, including many implicated in tumorigenesis, such as Raf-1, EGFR, v-Src family kinases, Cdk4, and ErbB-2 (Buchner J., 1999, TIBS, 24:136–141; Stepanova, L. et al., 1996, Genes Dev. 10:1491–502; Dai, K. et al., 1996, J. Biol. Chem. 271:22030–4).
In vivo and in vitro studies indicate that without the aid of co-chaperones HSP90 is unable to fold or activate proteins. For steroid receptor conformation and association in vitro, HSP90 requires Hsp70 and p60/Hop/Sti1 (Caplan, A., 1999, Trends in Cell Biol., 9: 262–68). In vivo HSP90 may interact with HSP70 and its co-chaperones. Other co-chaperones associated with HSP90s in higher eukaryotes include Hip, Bag1, HSP40/Hdj2/Hsj1, Immunophillins, p23, and p50 (Caplan, A. supra).
Many ansamycins, such as herbimycin A (HA) and geldanamycin (GM), bind tightly to a pocket in the HSP90s (Stebbins, C. et al., 1997, Cell, 89:239–250). The binding of ansamycins to HSP90 has been reported to inhibit protein refolding and to cause the proteasome dependent degradation of a select group of cellular proteins (Sepp-Lorenzino, L., et al., 1995, J. Biol. Chem., 270:16580–16587; Whitesell, L. et al., 1994, Proc. Natl. Acad. Sci. USA, 91: 8324–8328).
The ansamycin-binding pocket in the N-terminus of Hsp90 is highly conserved and has weak homology to the ATP-binding site of DNA gyrase (Stebbins, C. et al., supra; Grenert, J. P. et al., 1997, J. Biol. Chem., 272:23843–50). This pocket has been reported to bind ATF and ADP with low affinity and to have weak ATPase activity (Proromou, C. et al., 1997, Cell, 90: 65–75; Panaretou, B. et al., 1998, EMBO J., 17: 4829–36). In vitro and in vivo studies indicate that occupancy of the pocket by ansamycins alters HSP90 function and inhibits protein refolding. At high concentrations, ansamycins have been reported to prevent binding of protein substrates to HSP90 (Scheibel, T., H. et al, 1999, Proc. Natl. Acad. Sci. USA 96:1297–302; Schulte, T. W. et al., 1995, J. Biol. Chem. 270:24585–8; Whitesell, L., et al., 1994, Proc. Natl. Acad. Sci USA 91:8324–8328). Alternatively, they have also been reported to inhibit the ATP-dependent release of chaperone-associated protein substrates (Schneider, C., L. et al., 1996, Proc. Natl. Acad. Sci. USA, 93:14536–41; Sepp-Lorenzino et al., 1995, J. Biol. Chem. 270:16580–16587). In both models, the unfolded substrates are degraded by a ubiquitin-dependent process in the proteasome (Schneider, C., L., supra; Sepp-Lorenzino, supra.) Therefore, ansamycins act as generalized inhibitors of HSP90 function or as agents that mimic or antagonize the regulatory effects of endogenous ligands that bind to the pocket.
In both tumor and nontransformed cells, binding of ansamycins to HSP90 has been reported to result in the degradation of a subset of signaling regulators. These include Raf (Schulte, T. W. et al., 1997, Biochem. Biophys. Res. Commun. 239:655–9; Schulte, T. W., et al., 1995, J. Biol Chem. 270:24585–8), nuclear steroid receptors (Segnitz, B., and U. Gehring. 1997, J Biol. Chem. 272:18694–18701; Smith, D. F. et al., 1995, Mol. Cell. Biol. 15:6804–12), v-src (Whitesell, L., et al., 1994, Proc. Natl. Acad. Sci. USA 91:8324–8328) and certain transmembrane tyrosine kinases (Sepp-Lorenzino, L. et al.,. 1995, J. Biol. Chem. 270:16580–16587) such as EGF receptor (EGFR) and Her2/Neu (Hartmann, F., et al., 1997, Int. J. Cancer 70:221–9; Miller, P. et al., 1994, Cancer Res. 54:2724–2730; Mimnaugh, E. G., et al., 1996, J. Biol. Chem. 271:22796–801; Schnur, R. et al., 1995, J. Med. Chem. 38:3806–3812). The ansamycin-induced loss of these proteins is said to lead to the selective disruption of certain regulatory pathways and results in growth arrest at specific phases of the cell cycle (Muise-Heimericks, R. C. et al., 1998, J. Biol Chem. 273:29864–72).
Combination therapy utilizing 17-AAG for the treatment of certain cancers has been discussed. For example, Nguyen, D. et al., 1999, Journal Thoracic and Cardiovascular Surgery, 118:908–915, report that concurrent treatment of non-small cell lung cancer cells in vitro with 17-AAG and Taxol® enhances the toxicity of paclitaxel.
Cyclin D in complex with Cdk4 or Cdk6 and cyclin E-Cdk2 phosphorylate the protein product of the retinoblatoma gene, Rb. Researchers have reported that the protein product of the Rb gene is a nuclear phosphoprotein, which arrests cells during the G1 phase of the cell cycle by repressing transcription of genes involved in the G1 to S phase transition (Weinberg, R. A., 1995, Cell, 81:323–330). Dephosphorylated Rb inhibits progression through late G1, in part, through its interaction with E2F transcription family members, which ultimately represses the transcription of E2F target genes Dyson, N., 1998, Genes Dev., 12: 2245–2262). Progressive phosphorylation of Rb by the cyclin-dependent kinases in mid to late G1 leads to dissociation of Rb from Rb-E2F complexes, allowing the expression of E2F target genes and entry into the S phase.
The retinoblastoma gene product is mutated in several tumor types, such as retinoblastoma, osteosarcoma and small-cell lung cancer. Research also indicates that in many additional human cancers the function of Rb is disrupted through neutralization by a binding protein, (e.g., the human papilloma virus-E7 protein in cervical carcinoma; Ishiji, T, 2000, J Dermatol., 27: 73–86) or deregulation of pathways ultimately responsible for its phosphorylation. Inactivation of the Rb pathway often results from perturbation of p16INK4a, Cyclin D1, and Cdk4.
Most cancer therapies are not successful with all types of cancers. For example, solid tumor types ultimately fail to respond to either radiation or chemotherapy, so there remains a need for cancer treatments which target specific cancer types. Further, the treatment of cancers and other cell proliferative diseases usually requires the use of cytotoxic agents, which have severe side effects. Therefore, a need exists for more effective treatments, especially those with fewer adverse side effects than currently available. The present invention satisfies these needs and provides related advantages as well. The present invention provides novel methods for treating cell proliferative disorders through the use of ansamycins in combination with cytotoxic agents.